Formation Of Fraxinus Mandshurica And The Role Of FmPHV Gene In Vitro Regeneration

In this study,we set the system of callus induction,propagation and adventitious bud regeneration in stem cambium were established with E mandshurica annual shoots as test materials.The key regulatory genes FmPHV and promoter were cloned for bioinformatics analysis.The effect of overexpression of FmPHV in regeneration was further determined by transgene and in combination with the expression characteristics of key genes in the regeneration process.This study laid a foundation for the study of the reproduction utilization and in vitro regeneration mechanism.The main results are as follows:1.Acquisition of cambium and cambium callus induction culture of F.mandshurica:(1)The study of factors influencing the cambium training:to study the IB A,IAA,sucrose osmotic pressure change and picloram,factors such as low temperature treatment of F.mandshurica stem cambium and the influence of callus induce,under the cold and high concentration of IB A solution double handle,cambium callus starting rate is as high as 83.0%and showed better than other treatment combinations of proliferation activity.The results showed that the culture methods of cambium cells were favorable for callus formation.(2)Cambium acquisition and establishment of culture technology:from may to July,the annual growth branches of ash were treated with 50mg/L IBA solution at 4℃ for 22h,then treated with 0.5mg/L IB A solution at 23-25℃ for 15min,and the bark was removed.After the bark was placed upside-down in MD201 medium for 7 days,the xylem was completely removed and the side near the phloem was placed on MD201 medium.Cambium callus was obtained after 7 days of dark culture.Through stress and change of osmotic pressure to separate phloem and cambium,the callus initiation rate of explants was up to 83.0%.The paraffin section showed that the callus originated from cambium,and the neutral red staining showed that both cambium cells and callus cells were rich in small vacuoles,which could be used as the morphological basis for the identification of stem cells.(3)The establishment of the cambium callus double successive transfer culture techniques:put the cambium callus contains the TSA and 6-BA in D1 culture medium,light intensity of 8080μmol·m2·s-1,develop into MD201 medium after 21 days,in the culture medium surface with drops of hormone-free WPM liquid medium(WB1),solid-liquid double-decker train 21 days,will receive the loose callus into liquid medium,every culture 7 days transgenerational.The culture materials can be put into MD201 to continue the secondary generation,or the liquid suspension culture of the secondary generation.2.Cambium adventitious bud differentiation of callus technology:the establishment of the WPM in a large number of elements and calcium salt for ordinary WPM medium twice,the rest of the components of normal G2 medium(3.5 mg/L WPM+6-BA+1 mg/L picloram TDZ+1 mg/L+ 0.5 mg/L TSA+7 g/L agar+20 g/L sucrose),light intensity,8080μmol·m2·s-1 train after 28 days cambium callus under the loose contact with the medium cell group clearance form bud primordium structure,differentiation rate was 3.3%.3.The clone obtained the PHV gene and named it FmPHV.The full length of FmPHV coding region is 2112bp,with a complete open reading frame,and the coding protein is composed of 703 amino acids.The results of the spatiotemporal expression of FmPHV gene showed that June was the month in which the overall expression of FmPHV was the highest among the ash plants,and the expression level of FmPHV in bud and cambium callus was high.The expression of FmPHV increased significantly under 4℃ and ABA stress.4.Acquisition of overexpressed FmPHV and analysis of the effect of FmPHV on bud regeneration.ProkII:PHV-GUS stable infection vector was constructed,the hypocotyl was transformed by agrobacteria-mediated method,and the transgenic plants with FmPHV overexpression were successfully obtained by regeneration medium(WPM+5.6g/L agar+30g/L sucrose+0.8mg/L TDZ,pH=5.8).The expression levels of key genes in the bud regeneration pathway of FmPHV overexpression were measured,and the results showed that after the high expression of FmPHV,the expression levels of its downstream MP(MONOPTEROS/AUXIN RESPONSE FACTOR5)were 63.9 times higher.At the same time,the instantaneous infection of pA7:PHV-GFP on seedling promoted the expression of auxin,cytokinin and key genes of bud regeneration,indicating the important role of FmPHV in plant development and bud regeneration pathway.5.FmPHV gene promoter sequence was cloned,the sequence length was 2357bp,and the promoter contained multiple components related to tissue site specific expression,hormone regulation response components,stress response components and other cis-acting components.The promoter vector Px-PHV-GUSp was constructed,and the transient infection and GUS of 21d seedling were carried out.The results showed that the FmPHV promoter was highly active in the areas where the growth was particularly vigorous,such as cotyledon base,basal part,mesophyll and mesophyll margin.